METTL3/miR-192-5p/RICTOR/mTOR通路在儿童支气管哮喘发病中的作用机制  

The mechanism of the METTL3/miR-192-5p/RICTOR/mTOR pathway in the pathogenesis of childhood bronchial asthma

作　　者：屈昌雪 郭素玉 刘怀彬 苏国德 卢太苓 孙逊 Qu Changrue;Guo Suyu;Liu Huaibin;Su Guode;Lu Tailing;Sun Xun(Second Department of Respiratory Medicine,the Affiliated Xuzhou Children's Hospital of Xuzhou Medical University,Xuzhou 221006,China)

机构地区：[1]徐州医科大学附属徐州儿童医院呼吸内二科,徐州221006

出　　处：《国际呼吸杂志》2024年第11期1312-1318,共7页International Journal of Respiration

基　　金：徐州市重点研发计划项目(KC22196)。

摘　　要：目的研究METTL3/miR-192-5p/RICTOR/哺乳动物雷帕霉素靶蛋白(mTOR)通路在儿童支气管哮喘(哮喘)发病中的作用机制。方法本研究为实验研究。采用非随机抽样的方法选取2023年1月至12月徐州医科大学附属徐州儿童医院收治的哮喘患儿60例作为哮喘组, 另选择同期健康体检的60名儿童作为对照组。比较2组儿童CD4+ T淋巴细胞中METTL3 mRNA、RICTOR mRNA和miR-192-5p水平;流式细胞术检测并比较2组儿童Th1细胞、Th2细胞和Th1/Th2水平;定量检测并比较2组儿童CD4+ T淋巴细胞中N6-甲基腺苷(m6A)修饰水平;通过双荧光素酶报告基因实验验证miR-192-5p与RICTOR的靶向性, 建立4个实验组:RICTOR野生型组(转染RICTOR野生型载体)、RICTOR野生型+miR-192-5p过表达组(转染RICTOR野生型载体和miR-192-5p mimic质粒)、RICTOR突变型组(转染RICTOR突变型载体)和RICTOR突变型+miR-192-5p过表达组(转染RICTOR突变型载体和miR-192-5p mimic质粒)。将哮喘组患儿的CD4+ T淋巴细胞随机分为3组:无处理对照组(未进行任何转染处理)、NC mimic组(转染对照质粒NC mimic)和miR-192-5p过表达组(转染miR-192-5p mimic质粒)。蛋白质印迹法检测3组中RICTOR、mTOR蛋白水平, 并比较3组Th1细胞、Th2细胞和Th1/Th2水平。结果哮喘组患儿METTL3 mRNA和miR-192-5p水平均低于对照组, RICTOR mRNA水平高于对照组[(1.00±0.18)比(2.28±0.42), (1.00±0.23)比(2.74±0.45), (2.52±0.41)比(1.00±0.20), 均P<0.001]。哮喘组患儿Th1细胞、Th1/Th2水平均低于对照组, 而Th2细胞高于对照组[(4.12±0.23)%比(8.24±1.14)%, (1.35±0.45)比(3.81±1.08), (2.86±0.82)%比(1.97±0.58)%, 均P<0.001]。哮喘组患儿CD4+ T淋巴细胞中m6A修饰水平低于对照组[(0.24±0.07)%比(0.58±0.12)%, t=18.96, P<0.001]。RICTOR野生型+miR-192-5p过表达组的相对荧光素酶活性低于RICTOR野生型组[(0.41±0.07)比(1.05±0.12), t=7.98, P=0.001], RICTOR突变型+miR-192-5p过表达组与RICTOR突变型组的相对荧光�Objective:To explore the mechanism of the methyltransferase-like 3(METTL3)/miR-192-5p/rapamycin-insensitive companion of mammalian target of rapamycin(RICTOR)/mammalian target of rapamycin(mTOR)pathway in the pathogenesis of childhood bronchial asthma(asthma).Methods:This was an experimental study.A non-random sampling method was used to select 60 asthmatic children treated in the Affiliated Xuzhou Children′s Hospital of Xuzhou Medical University from January to December 2023 as the asthma group,and another 60 healthy children who underwent physical examination during the same period were selected as the control group.The mRNA levels of METTL3,RICTOR and miR-192-5p in CD4+T lymphocytes of children were compared.Flow cytometry was used to detect the levels of Th1 cells,Th2 cells,and Th1/Th2 in the two groups of children.Quantitative detection and comparison of N6-methyladenosine(m6A)modification levels in CD4+T lymphocytes of children were conducted.The targeting of miR-192-5p and RICTOR was verified through dual-luciferase reporter gene assay by quantifying luciferase activity in cells of RICTOR wild-type group(transfected with RICTOR wild-type vector),RICTOR wild-type+miR-192-5p overexpression group(transfected with RICTOR wild-type vector and miR-192-5p mimic plasmid),RICTOR mutant group(transfected with RICTOR mutant vector),and RICTOR mutant+miR-192-5p overexpression group(transfected with RICTOR mutant vector and miR-192-5p mimic plasmid).The CD4+T lymphocytes of the asthmatic children were randomly divided into three groups:the untreated control group(without any transfection treatment),the NC mimic group(transfected with the control plasmid NC mimic),and the miR-192-5p overexpression group(transfected with miR-192-5p mimic plasmid).Western blotting was used to detect the protein levels of RICTOR and mTOR in the three groups,and the levels of Th1 cells,Th2 cells,and Th1/Th2 were compared among the three groups.Results:Asthma group children had significantly lower mRNA levels of METTL3 and miR-192-5p compa

关 键 词：哮喘 儿童 N6-甲基腺苷 微RNAS 哺乳动物雷帕霉素靶蛋白 TH2细胞 

分 类 号：R725.6[医药卫生—儿科]